The goal of this proposal is to design new approaches and bench-top devices that enable high throughput separation and purification of exosomes from minimally processed patient serum samples. The aim is to produce highly purified materials that are devoid of genetic, proteomic, or lipidomic moieties exogenous to exosomal structures. Exosomes are natural biocompatible nanovesicles whose in vivo function is poorly understood given the difficulty of isolating large quantities of uniformly disperse exosome subpopulations for experimentation. In spite of this hurdle, new efforts have begun to focus on the high potential utility of exosomes as therapeutic nano-carriers. In contrast to artificial nanovesicles, cell-derived exosomes express a plethora of complex targeting ligands including integrins, receptors or even tetraspanins in native conformations and contain a cytoskeleton allowing for deformability. Consequently, exosomes are showing promise as cancer vaccines and have even been modified to deliver siRNA across the blood brain barrier. For these reasons, investigating means to study exosome function in vivo for the purposes of understanding their role in disease pathogenesis or adapt exosomes for use as therapeutic delivery vehicles is warranted. We are proposing to develop an instrument for separation and purification of exosomes without any labels such that these nanovesicles can be collected, modified and used as drug carriers without affecting their inherent biocompatibility and functionality. This work will employ field-flow fractionation (FFF) and split-flow lateral thin transport (SPLITT) techniques to characterize and continuously separate exosomes from cell cultures and identified serum and/or plasma samples. PI Gale, with expertise in FFF and microfluidics, has teamed up with co-PIs Wickline and Hood who are experts in exosome biology, to develop an instrument capable of characterizing exosomes based on biophysical properties and continuously separate exosomes without any labels or any structural/functional damage. The following are the specific aims for this project: Specific Aim 1: To characterize exosomes and identify optimal separation conditions using FFF. Specific Aim 2: To fabricate and test SPLITT and serial SPLITT systems for continuous exosome separation. Specific Aim 3: To demonstrate continuous separation of cultured and serum and/or plasma exosomes with serial SPLITT system.